The First Complete 3D Reconstruction of a Spanish Fly Primary Larva (Lytta vesicatoria, Meloidae, Coleoptera) Si-Qin Ge1,2, Benjamin Wipfler1,2, Hans Pohl2, Yi Hua1, Adam S´ lipin´ ski3, Xing-Ke Yang1*, Rolf Georg Beutel2 1 Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, China, 2 Entomology Group, Institut fu¨r Spezielle Zoologie and Evolutionsbiologie mit Phyletischem Museum, Friedrich-Schiller-Universita¨t Jena, Jena, Germany, 3 CSIRO Ecosystem Sciences, Australian National Insect Collection, Canberra, Australia Abstract The first detailed anatomical study of a primary larva of Meloidae is presented. Thereby techniques such as three- dimensional reconstructions, microtome sections, SEM (scanning electronic microscopy) and CLSM (confocal laser scanning microscopy) are applied. The structural features are discussed in the context of phylogeny, but also possible correlations with parasitism, phoresy and miniaturisation. The triungulin first instar larva is likely an apomorphy of Meloidae excl. Eleticinae and linked with a specialisation on acridoid eggs or larvae and provisions of bees. The campodeid body shape of Lytta and Meloinae is a groundplan feature of Meloidae, whereas a navicular body is an autapomorphy of the generally phoretic larvae of Nemognathinae. Head structures of Lytta and features of the postcephalic body are largely plesiomorphic. The musculature of the head is only moderately simplified while the one of the postcephalic body is well developed. Its thorax is largely characterised by plesiomorphies. The characteristics of the legs suggest phoretic habits, even though this does not apply to larvae of Lytta. It is conceivable that a phoretic behaviour is secondarily lost, together with some but not all morphological modifications related to it. Derived features of the abdomen of Meloidae are the complete loss of the fixed urogomphi (also missing in Rhipiphoridae and other related groups) and the presence of one or two conspicuous caudal bristles. Only few features of Lytta are shared with the parasitic larvae of Rhipiphoridae and Strepsiptera. These characteristics, which are possibly linked with specialised life habits, have obviously evolved independently. Miniaturisation effects are minimal in the larvae of Lytta. Citation: Ge S-Q, Wipfler B, Pohl H, Hua Y, S´lipin´ski A, et al. (2012) The First Complete 3D Reconstruction of a Spanish Fly Primary Larva (Lytta vesicatoria, Meloidae, Coleoptera). PLoS ONE 7(12): e52511. doi:10.1371/journal.pone.0052511 Editor: Andreas Hejnol, Sars International Centre for Marine Molecular Biology, Norway Received September 26, 2012; Accepted November 14, 2012; Published December 26, 2012 Copyright: ß 2012 Ge et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The project partly supported by grants from the National Science Foundation of China (Grant Nos. 31010103913 and 30970390), the Knowledge Innovation Program of Chinese Academy of Sciences (No. KSCX3-IOZ-1004), the National Science Fund for Fostering Talents in Basic Research (Special Subjects in Animal Taxonomy, NSFC-J0630964/J0109, J0930004). BW is supported by the DFG (grant number BE1789/61) and the Chinese Academy of Sciences Fellowshipfor Young International Scientist (grant number 2011Y2SB05). The funders had no role in study design, data collection and analysis, decision to publish,or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction prominent figures like for instance Livia, the wife of the Roman emperor Augustus, the German Emperor Henry IV, or the Lytta vesicatoria (Linnaeus) is a charismatic species of the medium Marquis de Sade. Irritation of urethral passages and inflammation sized tenebrionoid family Meloidae, which comprises ca. of the genitalia can lead to painful priapism. In the 19th century 3000 spp. worldwide (e.g. [1;2]). The Meloidae group occurs on cantharidin was one of the most poisonous known substances and all continents with the exception of Antarctica but is absent from only as little as 10 mg can be lethal for humans. In ancient China, New Zealand and most Polynesian islands. Lytta vesicatoria is a fairly the beetles were mixed with excrement, arsenic and wolfsbane to large and conspicuous beetle with a metallic emerald-green create the world’s first recorded stink bomb. coloration. It occurs mainly in the Mediterranean area but Another remarkable feature of Lytta and the other meloids (with occasionally it is also collected in Central or Eastern Europe and the possible exception of Eleticinae; [2]) is a specific type of Asia The adults are usually found exposed on its food plants, parasitism, linked with hypermetamorphosis, and miniaturised especially Fraxinus excelsior (Common Ash) and F. ornus (Manna triungulin first instar larvae. The tringulins of many meloid groups Ash), but also on poplar, elderberry, lilac, olive and others. Like in (Meloinae partim, Nemognathinae [excl. Stenodera] are phoretic other ‘‘blister beetles’’ (Meloidae) males produce cantharidin, and attach to their bee hosts as they visit flowers [2;4]. This is not a vesicating substance functioning as a very efficient and powerful the case in Lytta [5] even though they seem to be well adapted for defensive natural product [2]. Cantharidin is transmitted during such a behaviour (e.g., triungulin legs). The hypermetamorphic copulation and placed on the egg surface by the females. The development passes through eight immature stages, the tringulin, powerful terpenoid can cause blisters and seriously affect the the first grub (instars II–V), the coarctate (instar VI), the second gastrointestinal and urinary tract, and also the kidneys (e.g. [3]). grub (instar VII), and the pupa. After attaining the host’s nest the Historically it is well known as an aphrodisiac, supposedly used by triungulin starts to feed on the bee’s food-stores and eventually PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e52511 Morphology of the Larva of Lytta vesicatoria molts [2;4]. The four stages of the relatively immobile but feeding figures were prepared with Photoshop CS5 (Adobe) and Illustrator scarabaeiform first grub is followed by the inactive coarctate, CS5 (Adobe). which is characterized by a diapause. Then follows the active but non-feeding second grub, which is morphologically similar to the Scanning Electronic Microscopy (SEM) first grub and finally prepares the pupal chamber. Specimens were transferred to 100% ethanol, then dried at the The focus of the present study is on the agile first instar larvae, critical point (Emitech K850 critical point dryer) and subsequently the stage which is crucial for the access to the host’s resources (or sputter-coated (Emitech K500). Microscopy was performed on body) as it is the case in different ways in several other parasitic a Philips XL30 ESEM using a special specimen holder [19]. groups of insects (e.g., Brachininae [Carabidae], Rhipiphoridae, Passandridae, Bothrideridae, Strepsiptera; e.g., [6;7]). The prima- Confocal Laser Scanning Microscopy (CLSM) ry aim of our investigation is the first full documentation of the For CLSM specimens were mounted between cover glass and external and internal morphology of a tenebrionoid triungulin glass slide in a drop of ethanol. Image stacks were created with larva. Generally anatomical information on beetle larvae is a Zeiss LSM 510 using the auto fluorescence at 488 nm of argon relatively scarce. The head morphology of some groups is laser. comparatively well known through a series of studies published in the two last decades (e.g. [8;9;10;11]), whereas internal Muscular Nomenclature structures of the postcephalic body are largely unknown. The The muscular terminology follows Wipfler et al. [20] and v. presently available anatomical knowledge is restricted to a study Ke´ler [21] (in bracelets) for the head and v. Ke´ler [21] for the focussed on the nervous system of the mealworm Tenebrio molitor abdomen. The thoracic muscles are consecutively numbered and [12], one on an extremely miniaturised ptiliid larva [13], and one do not follow any previously used nomenclature. on the larva of the corylophid Sericoderus [14]. The morphological findings will be discussed with respect to the unclear phylogenetic Results affinities of Meloidae within Tenebrionoidea, and the position of Lytta within the family. Another aim was to evaluate effects of 1 General Appearance and Differences between Instars miniaturisation in the small and slender triungulin larvae, The campodeiform first instar larvae are distinctly sclerotized following a series of recent studies on this topic using various triungulins with a slender, subparallel body and slender legs. They groups of beetles but also other groups of insects (e.g. are slightly constricted at the metathorax and tapering at the [13;14;15;16]). The third aim is to evaluate effects of a parasitic terminal abdominal segments VIII and IX (Fig. 1). The body of lifestyle on the morphology of the triungulins, also focussing on second instar larvae is very lightly sclerotized and scarabaeiform. parallelisms with other parasitic groups with hypermetamorphosis, The antennae and eyes are reduced. Legs are present but shorter such as for instance Rhipiphoridae